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- Gonzalez-Castrillon, Luz Maria, 1990-
(author)
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Molecular mechanisms of nerve-tumor interactions : the intersection of cancer and neurodevelopment
- 2023
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Doctoral thesis (other academic/artistic)abstract
- A wide range of cancers throughout the body are characterized by high nerve density and invasion of cancer cells within the nerves, a process called perineural invasion (PNI). Work in the field has shown that blocking nerves in organs with tumors leads to improved disease outcomes suggesting that finding ways to block tumor nerves could lead to new treatment approaches. Despite the importance of this, little is known about the molecular mechanisms underlying nerve-tumor interactions. An increasing number of studies have revealed that the genes associated with PNI are classical neurodevelopmental genes associated with neurodevelopmental processes. Therefore, the central hypothesis of this thesis was that nerve-tumor interactions result in part from an abnormal reactivation of the molecular pathways underlying the embryonic development of the nervous system. To test this hypothesis, public datasets from different types of cancer with high incidence of PNI were analyzed to identify molecular pathways common between these cancers. This analysis revealed that neurodevelopmental pathways accounted for 12 - 16% of the differentially expressed genes (DEGs), with axon guidance genes being markedly dysregulated. Overall, 17 different axon guidance gene families, including ephrin-Eph, semaphorin-neuropilin/plexin and slit-robo pathways were dysregulated. Further disruption of these pathways was a common feature across a number of cancers analyzed and their dysregulation had a significant impact on disease survival. Overall, this suggested that neurodevelopmental molecular pathways may contribute to tumor axonogenesis and PNI. These findings suggested a significant role of neurodevelopmental pathways in cancer dysregulation. Thus, a comprehensive understanding of the pathways during the nervous system development is imperative. Therefore, in my thesis, the embryo was used as a tool to study the mechanisms by which these molecular pathways influence axonogenesis more broadly. First, the role of the axon guidance genes Slit/Robo was examined during mouse neurodevelopment. Our results showed that Robo2 enrichment influences the migration and axonal projections of spinal ipsilateral neurons. In parallel, we investigated the role of alternative splicing of transcription factors as a mechanism of increase neuronal diversity. In particular we examined the expression dynamics of Lhx9, a transcription factor that controls the expression of the axon guidance gene Robo3. Lhx9 splice variants showed a differential expression at key developmental points in the spinal cord, suggesting Lhx9 splice dynamics plays an important role in neural guidance choices. In the third part of the thesis, I investigated the role of gap junctions, in nerve-tumor interactions, using pancreatic ductal adenocarcinoma (PDAC) cancer cells in vitro models. The connexin GJB2 emerged as the most overexpressed gap junction component in PDAC tumors. In vitro analysis, involving blocking gap junctions or connexin overexpression, revealed that gap junctions influenced PDAC cancer cell behaviors and properties. Further we developed a novel nerve-tumor assay and used it to examine the role of gap junction genes in PDAC cells neuronal growth. Overall, this thesis postulated that several key molecular pathways crucial for normal nervous system embryonic development, could underly nerve- tumor interactions during cancer development and progression.
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| 2. |
- Pu, Longjun, 1990-
(author)
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A molecular exploration of sensory responses in c. elegans
- 2023
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Doctoral thesis (other academic/artistic)abstract
- Sensation provides a pivotal ability, allowing animals to survive in complex environments. The cues sensed by animals are represented by external stimuli and internal signals. However, the mechanisms mediating sensations in molecular and cellular level are still not well-studied. In this thesis, by using free-living nematodes C. elegans with relatively simple nerve system, we are trying to get better understandings of molecular mechanisms by which animals sense and interpret external cues and internal signals.G protein-coupled receptors (GPCRs), as one of the major families of transmembrane proteins, participate in a variety of physiological responses to both external stimuli and internal cues. Previous studies have shown that GPCR signals are broadly involved in many processes in C. elegans, such as olfactory sensing, nociceptive responses, social behavior, pathogen responses, and mating. However, the complexity and diversity of GPCRs pose significant challenges to systematic dissection of their function as well as identification of receptor-ligand pairs which play crucial roles for animals´ sensory behaviors. Interestingly, the genome of C. elegans encodes one of the largest GPCR repertoires among any sequenced organisms, indicating a dramatical expansion and high degree of gene redundancy. To comprehensively dissect GPCR signaling in C. elegans and gain more insights into their roles in sensations, we developed an approach by employing CRISPR/Cas9-based gene editing to mutate closely related GPCRs and neuropeptide genes (internal signals) in a single strain on a genome-wide scale, resulting in disrupting nearly all the GPCR and neuropeptide genes (more than 1800 genes in total) and eliminating high degree of gene redundancy as well. Then using these two genetic libraries, we successfully identified neuropeptide (FLP -1) and cognate receptors (DMSR-4, DMSR-7 and DMSR-8) required for hypoxia-evoked locomotory responses, obtained a set of novel regulators of the pathogen-induced immune response including FMI-1 and DOP-6, and especially identified receptors (SRX-64) in AWA neurons for the volatile odorant pyrazine and redundant receptors (SRX-1, SRX-2 and SRX-3) in AWCOFF neuron for 2,3-pentanedione.In nature, animals often experience and sense constantly changing gas environments. And human bodies also generate internal gas as gasotransmitters for signal transduction, such as CO, NO and H2S. For the mechanism governing sensory and adaptive responses to different gaseous cues, extensive studies are still needed. Here, taking advantage of the robust locomotory responses to H2S in C. elegans, we delineated the molecular mechanisms of H2S sensation and adaptation. We found that C. elegans exhibited transiently increased locomotory and turning activity as a strategy to escape the noxious H2S. The behavioral responses to H2S were modulated by a complex network of signaling pathways, ranging from cyclic GMP signaling in ciliated sensory neurons, calcineurin, nuclear hormone receptors, to the major starvation regulators such as insulin and TGF-β signaling. Prolonged exposure to H2S robustly evoked H2S detoxification and reprogrammed gene expression, where genes involved in iron homeostasis, including ftn-1 and smf-3, were robustly modified, implying that labile iron levels are affected by H2S. In addition, the roles of labile iron for modulating H2S response were further investigated by using genetic studies and chemical applications. Interestingly, the response to H2S was substantially affected by the ambient O2 levels and their prior experience in low O2 environments, suggesting an intricate interplay between O2 and H2S sensing. The crosstalk is often seen between different experiences and sensations. In addition to the interplay between O2 and H2S sensing, we found hypoxia challenge could induce food leaving behavior in C. elegans. The alteration of food behavior by hypoxia experience was independent of the known mechanisms involved in O2 response, including pathways in acute hypoxia and HIF-1 signaling for chronic hypoxia response. The robust failure of induced food avoidance in egl-3 and egl-21 mutants suggested that neuropeptidergic signaling was required for this response. And future work is needed for comprehensively understanding the roles of neuropeptide signaling in the crosstalk between hypoxia experience and food leaving behavior.In summary, our studies shed light on the molecular and cellular mechanisms of how animals sense and interpret the signals, allowing them to survive in a complex environment niche. More specifically, 1) we demonstrated the dissection of genetic landscape of GPCR signaling through phenotypic profiling in C. elegans. And as a powerful genetic resource, our libraries can greatly expedite the analyses of GPCR signaling in multiple additional contexts. 2) we provided molecular insights into how C. elegans detects and adapts its response to H2S and modulates behaviors through ambient environment and experience.
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| 3. |
- Tadala, Lalitha, 1991-
(author)
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Role of extracellular ATP in immune mechanisms against infections
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Inflammation is driven either by infection with pathogens or sterile stimuli, such as tissue damage or autoimmune diseases. Upon tissue damage, ATP is released passively from the dead or compromised cells. During stress, ATP can be secreted from the cells. Extracellular ATP (eATP) acts as an endogenous danger signal. An increase in eATP is sensed by cell surface purinergic receptors and regulates the onset and resolution of inflammation. Extracellular ATP is an important inflammatory mediator during sterile inflammation. On the other hand, the role of eATP is poorly studied during infection, both bacterial and viral. In this thesis, I present the molecular mechanisms underlying ATP secretion during bacterial infections and the role of eATP in human hantaviral infections.During infection with certain enteropathogenic Gram-negative bacteria, intestinal epithelial cells secrete ATP via connexin hemichannels as an alert signal to activate the immune system, which triggers acute inflammation in the gut. However, neither what triggers ATP secretion nor the molecular mechanisms of ATP secretion were known. Pharmacological, genetic, and microscopy-based evidence shows that during invasive bacterial infections, the plasma membrane ruffles act as mechanical immune stimuli and activate the inherently mechanosensitive plasma membrane channel PIEZO1. Mechanically activated PIEZO1 leads to the influx of Ca2+ ions and concurrent ATP secretion. In addition, PIEZO1 also activates protective transcriptional responses. Thus, PIEZO1 acts as a sensor for invasive infection using mechanical stimuli, unlike the so-far-described immune sensors of infection, which all recognize microbial components by chemical interaction.During human hantavirus infection, the humoral immune responses are poorly studied. Our collaborators found that atypical B cells, which do not have the surface marker CD27, show increased frequency in a cohort of hantavirus-infected patients. CD27 shedding in murine lymphocytes had been previously linked to eATP-dependent activation of a purinergic receptor7. To test whether ATP levels in the circulation of hantavirus-infected patients are elevated, an approach to perform same-day eATP quantifications in human plasma was developed. This assay was used to establish the normal eATP concentration in plasma in a cohort of healthy volunteers and to show that eATP levels are elevated in the acute and convalescent stages of hantavirus infection. Further, the addition of ATP to isolated human B cells recapitulated the observed CD27 shedding via a metallomatrix proteinase-8-dependent (MMP8) mechanism. Together, these projects provide evidence for the importance of eATP in bacterial and viral infectious diseases.
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